Fig. 6: RAD51C KO and V239G complemented cell lines have impaired replication fork reversal resulting in ssDNA gap formation.

a Schematic of labeling of replication fork progression. The cells were first pulsed with CldU for 20 min followed by IdU with camptothecin (100 nM CPT) for 60 min subsequently medium with or without the S1 nuclease was added for 20 min. Replication fork speed in untreated cells was assessed in Supplementary Fig. 4. b RAD51C knockout cells and RAD51C-V239G expressing cells exhibit increased DNA tract lengths and increased ssDNA gaps. RAD51C CRISPR/Cas9 U2OS cells were complemented with the WT RAD51C or the indicated variant and DNA replication fork progression was determined. Each data point represents Idu/Cldu ratio for one fiber measurement. Approximately 200 fibers were analyzed from two experiments and bars represent median of each data set. Statistical significance was determined by Kruskal-Wallis with multiple Dunn’s comparison and is indicated (ns is not significant, ** p = 0.0071, *** p = 0.0034, **** p < 0.0001). From left to right, n = 204, 201, 203, 201, 203, 206, 202, 203, 203, or 204 fibers quantified between two biological replicates. c RAD51C variants uncouple the enzymatic, HR, and replicative functions. Summary of phenotypes of RAD51C variants V239G and L238R and A279P from Figs. 1–5. Green is indicative of wild-type RAD51C function, yellow is indicative of a partial function, and red is indicative of a reduced function. Note that the DNA binding and ATPase activities were analyzed for V239G and A279P only since L238R formed insoluble aggregates. Source data are provided as a Source Data file.